It is particularly preferred to employ Staphylococcal genes and gene products as targets for the development of antibiotics. The Staphylococci make up a medically important genera of microbes. They are known to produce two types of disease, invasive and toxigenic. Invasive infections are characterized generally by abscess formation affecting both skin surfaces and deep tissues. S. aureus is the second leading cause of bacteremia in cancer patients. Osteomyelitis, septic arthritis, septic thrombophlebitis and acute bacterial endocarditis are also relatively common. There are at least three clinical conditions resulting from the toxigenic properties of Staphylococci. The manifestation of these diseases result from the actions of exotoxins as opposed to tissue invasion and bacteremia. These conditions include: Staphylococcal food poisoning, scalded skin syndrome and toxic shock syndrome.
The frequency of Staphylococcus aureus infections has risen dramatically in the past few decades. This has been attributed to the emergence of multiply antibiotic resistant strains and an increasing population of people with weakened immune systems. It is no longer uncommon to isolate Staphylococcus aureus strains which are resistant to some or all of the standard antibiotics. This phenomenon has created a demand for both new anti-microbial agents, vaccines, and diagnostic tests for this organism.
The enzyme UDP-N-acetylmuramoyl-L-alanyl-D-glutamate:lysine ligase, encoded by the gene MurE, catalyses the addition of the third amino acid (meso-diaminopimelic acid or lysine) of the peptide moiety in peptidoglycan biosynthesis to form UDP-N-acetylmuramate tripeptide. The gene has been cloned and sequenced from Escherichia coli and the corresponding protein has been over-expressed, purified and characterised (Michaud, C., Mengin-Lecreulx, D., van Heijenoort, J. & Blanot, D. (1990) Eur. J. Biochem., 194, 853-861). MurE has also been sequenced from such organisms as Bacillus subtilis and Haemophilus influenzae.
The discovery of a MurE homologue in the human pathogen Staphylococcus aureus will allow us to produce UDP-N-acetylmuramoyl-L-alanyl-D-glutamate:lysine ligase enzyme, which can then be used to screen for novel inhibitors. Inhibitors of this protein have utility in antibacterial therapy as they will prevent the construction of the bacterial cell wall.
Clearly, there exists a need for factors, such as the MurE embodiments of the invention, that have a present benefit of being useful to screen compounds for antibiotic activity. Such factors are also useful to determine their role in pathogenesis of infection, dysfunction and disease. There is also a need for identification and characterization of such factors and their antagonists and agonists to find ways to prevent, ameliorate or correct such infection, dysfunction and disease.
Certain of the polypeptides of the invention possess amino acid sequence homology to a known MurE from Bacillus subtilis protein. See Swissprot Accession No. Q03523. Also see Michaud, C., Mengin-Lecreulx, D., van Heijenoort, J. & Blanot, D. (1990) Eur. J. Biochem., 194, 853-861; DANIEL R. A., ERRINGTON J., J. GEN. MICROBIOL. 139:361-370 (1993); and DANIEL R. A., DRAKE S., BUCHANAN C. E., SCHOLLE R, ERRINGTON J.; J. MOL. BIOL. 235:209-220 (1994).